When monochromatic light such as laser light strikes a sample, almost all of the light is scattered elastically, which is called Rayleigh scattering. This Rayleigh scattered light undergoes no change in energy or frequency. However, a very small portion of the light, xcx9c1 in 108, is scattered in elastically, which is called Raman scattering. This light does undergo a change in energy and frequency, and the change corresponds to an excitation of the illuminated molecular system, most often excitation of vibrational modes. Measuring the intensity of the Raman scattered photons as a function of the frequency difference provides a Raman spectrum. Raman peaks are typically narrow (a few wavenumbers) and in many cases can be associated with the vibration of a specific chemical bond (or normal mode dominated by the vibration of a single functional group) in a molecule. As such, it is a xe2x80x9cfingerprintxe2x80x9d for the presence of various molecular species and can be used for both qualitative identification and quantitative determination. Analytical Raman Spectroscopy, Chemical Analysis Series Vol. 114, J. G. Grasselli and B. J. Bulkin, Eds. (John Wiley, New York, 1991).
Raman spectroscopy has a variety of potential uses in vivo. For example, Raman spectra have been observed from various biological tissues including skin. Ozaki, Y.: xe2x80x9cMedical application of Raman spectroscopyxe2x80x9d Appl. Spectr. Rev. 24:259-312, 1988; Manoharan, R., et al. xe2x80x9cHistochemical analysis of biological tissues using Raman spectroscopy,xe2x80x9d Spectro. Acta Part A, 52:215-249, 1996; Mahadevan-Jansen, A. and Richards-Kortum, R.: xe2x80x9cRaman spectroscopy for the detection of cancers and precancers,xe2x80x9d J. of Biomed. Op., 1:31-70, 1996. Identified Raman scatterers in tissues include elastin, collagen, blood, lipid, tryptophan, tyrosine, carotenoid, myoglobin. Id. Most of the data has been obtained from ex vivo tissue samples using Fourier-Transform (FT) Raman spectrometers. These data have demonstrated that Raman spectroscopy has potential for diagnosis of diseases. Raman spectroscopy might also be used to monitor cutaneous drug delivery and pharmacokinetics during skin disease treatment. Schallreuter, K. U.: xe2x80x9cSuccessful treatment of oxidative stress in vitiligo,xe2x80x9d Skin Pharmacol. Appl. Skin Physiol. 12(3):132-8, 1999; Lawson, E. E., et al. xe2x80x9cInteraction of salicylic acid with verrucae assessed by FT-Raman spectroscopy,xe2x80x9d J. Drug Target 5(5):343-51, 1998; Schallreuter, K. U., et al. xe2x80x9cIn vivo evidence for compromised phenylalanine metabolism in vitiligo,xe2x80x9d Biochem. Biophys. Res. Commun. 13;243(2):395-9, 1998; Schallreuter, K. U., et al. xe2x80x9cOxybenzone oxidation following solar irradiation of skin: photoprotection versus antioxidant inactivation,xe2x80x9d J. Invest. Dermatol. 106(3):583-6, 1996. Raman spectroscopy could also potentially be used to detect the presence of prohibited drugs used by athletes or specific drugs in drug abusers.
In order to enhance such uses, Raman measurements should be able to be performed in vivo and quickly, preferably within seconds or sub-seconds. FT-Raman systems typically require as much as half an hour to acquire a spectrum and are typically bulky and not portable. Manoharan, R., et al. xe2x80x9cHistochemical analysis of biological tissues using Raman spectroscopy,xe2x80x9d Spectro. Acta Part A, 52:215-249, 1996. Recently developed dispersive-type Raman systems based on fiber optic light delivery and collection, compact diode lasers, and high efficiency spectrograph-detector combinations, may be able to acquire in vivo tissue Raman spectrum in seconds. Baraga, J. J., et al. xe2x80x9cRapid Near-infrared Raman spectroscopy of human tissue with a spectrograph and CCD detector,xe2x80x9d Appl. Spectro. 46:187-90, 1992; Kramer, J. R., et al. xe2x80x9cSpectral diagnosis of human coronary artery: a clinical system for real time analysis,xe2x80x9d SPIE Proc. 2395:376-82, 1995; Mahadevan-Jansen, A., et al. xe2x80x9cDevelopment of a fiber optic probe to measure NIR Raman spectra of cervical tissue in vivo,xe2x80x9d Photochem. Photobiol. 68(3):427-31, 1998.
Thus, there is a need for Raman spectroscopy systems capable of fast speeds or high quality results. The present invention provides these and other advantages.
The present invention provides systems and methods for rapid Raman spectroscopy. The speed is improved by providing light from a sample to a light-dispersive element, such as a holographic grating, in a pattern that inversely complements distortion caused by the grating or other device. For example, if the grating imparts a curve to the spectral lines emanating from the grating, then the light is inserted into the grating in a curve in the opposite direction. These and other features of the present invention enhance the signal to noise ratio and improve the spectral resolution of the system. The present invention also provides a calibration light guide able to transmit a known, or standard, light to the detection or spectroscopy system. The calibration light guide can be useful both with traditional light transmission guides and with the light transmission guides of the present invention.
In one aspect, the present invention provides a light transmission bundle suitable for use for Raman spectroscopy, the light transmission bundle comprising a proximal end and a distal end and comprising at least 5 light guides, wherein the light guides are arranged in a substantially filled-in geometrical shape at the proximal end of the light transmission bundle and a substantially linear curve at the distal end. In some embodiments, the substantially linear curve is a parabolic curve, and can be substantially identical to a curve of a substantially linear line of light after it has been passed through a holographic grating, for example a volume phase technology (VPT) holographic grating. In some embodiments, the filled-in geometrical shape is a circle, although other shapes are also possible. (Unless expressly stated otherwise or clear from the context, all embodiments of the present invention can be mixed and matched.)
The present invention also provides a calibration light guide, which can be one of the light guides at the distal end of the light transmission bundle described above, for example at the center of the substantially linear curve. Typically, the proximal end of the calibration light guide is optically connected to a calibration light source.
In another aspect, the present invention provides a Raman spectrometer system comprising a detection light guide able to detect light emanating from a sample. A distal end of the detection light guide is optically connected to a plane grating that is in turn optically connected to a pixelated light detector operably connected to a controller containing computer-implemented programming that detects light impinging on detection pixels in the pixelated light detector. A light transmissive portion of the distal end of the detection light guide is arranged in a substantially inverse shape that is complementary to a distortion to the light caused by passing the light through the plane grating, to provide light in a substantially straight line at the pixelated light detector. In some embodiments, the light guide is similar to that described above.
The Raman spectrometer system can further comprise a monochromatic illumination light source that provides illumination light to the sample, and an illumination light guide and a probe located at the distal end of the illumination light guide, wherein the illumination light guide transmits the monochromatic illumination light from the light source to the probe, which in turn transmits the illumination light to the sample. The monochromatic illumination light source can be an infrared laser, for example having a power of at least about 250 mW, a wavelength of about 785 nm, and a power of about 300 mW. The illumination light guide can be a single optical fiber having a diameter less than about 200 xcexcm. Preferably, the proximal end of the detection light guide is optically connected to the probe.
The probe can further comprise a compound parabolic concentrator (CPC) optically connected at the proximal end of the detection light guide, wherein the compound parabolic concentrator collects light emanating from the sample and concentrates it and transmits it into the distal end of detection light guide. Alternatively, the proximal end of the detection light bundle can have a large a diameter, for example greater than about 1 mm, and then the detection probe may not comprise a CPC. In some embodiments, the probe is sized to provide an illumination spot on the sample that is substantially larger than a detection spot detected by the probe.
The Raman spectrometer system can further comprise a calibration light guide optically connected to a calibration light source. Also, the detector preferably comprises an array of detection pixels and the detection light guide preferably comprises a bundle comprising enough light guides to substantially fill a column or row of the array, wherein the bundle comprises more than about 50 light guides. The light guides can be selected from the group consisting of an optical fiber, a liquid light guide and a hollow reflective light guide. Also, the system is preferably portable.
The present invention further provides means for and steps of achieving the various aspects, embodiments and features of such invention.
In a further aspect, the present invention provides methods of making a light transmission bundle comprising a) providing at least 5 light guides, b) arranging the light guides in a filled-in geometrical shape at a proximal end of the light transmission bundle, and c) arranging the light guides in a substantially linear curve at the distal end of the light transmission bundle. In some embodiments, the methods further comprise adding a calibration light guide to the light transmission bundle.
In another aspect, the present invention provides methods of taking a Raman measurement comprising a) illuminating a sample under conditions and for a time sufficient to induce measurable Raman scattered light to emanate from the sample, b) collecting the emanating light, c) providing the emanating light with a substantially inverse shape that is complementary to a distortion to the light caused by passing the light through a light-dispersive element, d) passing the light having the substantially inverse shape through the plane grating to provide substantially straight spectral lines, and e) performing Raman spectroscopic analysis on the substantially straight spectral lines. In some embodiments, providing the substantially inverse shape further comprises transmitting the emanating light through a detection light guide wherein the light transmissive portion of the distal end of the detection light guide is arranged in a substantially inverse shape that is complementary to a distortion to the light caused by passing the light through the light transmissive element, and wherein the light transmissive element comprises a plane grating. Illuminating the sample and collecting the emanating light can be done through a probe optically connected to an illumination light source and to the detection light guide, for example by providing an illumination spot on the sample that is substantially larger than the detection spot detected by the probe. The methods can further comprise providing calibration light, and can be performed using a portable Raman system, and in less than about 1 second.
These and other aspects of the present invention will become evident upon reference to the following Detailed Description and attached drawings. In addition, various references are set forth herein that describe in more detail certain apparatus and methods; all such references are incorporated herein by reference in their entirety.